Antimicrobial Activity of an Amazon Medicinal Plant (Chancapiedra) (Phyllanthus niruri L.) against Helicobacter pylori and Lactic Acid Bacteria

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1 PHYTOTHERAPY RESEARCH Phytother. Res. : 9 99 () Pulished online Octoer in Wiley Online Lirry (wileyonlinelirry.com) DOI:./ptr. Antimicroil Activity of n Amzon Medicinl Plnt (Chncpiedr) (Phyllnthus niruri L.) ginst Helicocter pylori nd Lctic Acid Bcteri Len Gálvez Rnill, Emmnouil Apostolidis nd Klids Shetty * Escuel de Alimentos, Fcultd de Recursos Nturles, Pontifici Universidd Ctólic de Vlpríso, Avenid Wddington, Ply Anch, Vlpríso, Chile Deprtment of Food Science, Chenoweth Lortory, University of Msschusetts, Amherst, MA, USA The potentil of wter extrcts of the Amzon medicinl plnt Chncpiedr (Phyllnthus niruri L.) from Ecudor nd Peru for ntimicroil ctivity ginst Helicocter pylori nd different strins of lctic cid cteri such s Lctocillus cidophilus, Lctocillus csei nd Lctocillus plntrum ws investigted. H. pylori ws inhiited y oth wter extrcts in dose dependent mnner, wheres lctic cid cteril growth ws not ffected. Both extrcts contined ellgic cid nd hydroxycinnmic cid derivtives nd exhiited high free rdicl scvenging linked-ntioxidnt ctivities (9%). However, gllic cid ws detected only in the Ecudorin extrct. Preliminry studies on the mode of ction of Chncpiedr ginst H. pylori reveled tht inhiition my not involve proline dehydrogense-sed oxidtive phosphoryltion inhiition ssocited with simple mono-phenolics nd could involve ellgitnnins or other non-phenolic compounds through yet unknown mechnism. This study provides evidence out the potentil of Chncpiedr for H. pylori inhiition without ffecting eneficil lctic cid cteri. Copyright John Wiley & Sons, Ltd. Keywords: Phyllnthus niruri L.; Helicocter pylori; Lctocillus; ntimicroils; phenolic ntioxidnts. INTRODUCTION Helicocter pylori is grm-negtive, microerophilic nd spirl-shped cterium tht hs een implicted in gstritis, peptic ulcer disese nd over long exposure potentilly to gstric crcinom (Mlfertheiner et l., ). H. pylori colonize the stomch of pproximtely hlf of the world s popultion nd unless specificlly treted, infection with this gstric pthogen is lifelong (Vorvuthikunchi nd Mitchell, ). H. pylori induces strong inflmmtory response within the host, therey relesing high level of host-derived toxic oxygen species, ut H. pylori cn survive nd colonize persistently in the hrsh conditions of the gstric mucos. To comt such forms of oxidtive stress, H. pylori expresses severl key iochemicl responses tht include the oxidtive stress resistnce enzymes superoxide dismutse nd ctlse, fmily of peroxiredoxins nd newly descried ntioxidnt proteins such s the NADPH quinone reductse (Wng et l., ). Tretment of H. pylori infection consists of multiple therpies tht comine wide rnge of ntiiotics such s tetrcycline, moxicillin, imidzoles nd few selected mcrolides (clrithromycin). However, the increse in ntiiotic resistnce nd host-ssocited side effects reduces the efficcy of such tretments (Kusters et l., ). In view of the incomplete cure chieved * Correspondence to: K. Shetty, Deprtment of Food Science, Chenoweth Lortory, University of Msschusetts, Amherst, MA, USA. E-mil: klids@foodsci.umss.edu Copyright John Wiley & Sons, Ltd. with ntiiotic-sed therpy due to resistnt strins, investigtions on nturlly occurring ntimicroil compounds found in dietry, medicinl plnts nd her extrcts s lterntive nd/or s ioctive gents supporting ntiiotics for treting H. pylori infection hve excellent potentil. Phyllnthus niruri L. (Euphoricee) is smll erect nnul her growing up to cm in height nd is indigenous to the Amzon rinforest nd other tropicl res including South Est Asi, Southern Indi nd Chin (Girch et l., 99). This plnt hs long history in herl medicine systems nd hs een used widely in different regions of the world for the tretment of vrious diseses. In vitro studies hve shown tht wter extrcts of P. niruri L. inhiit the clcium oxlte crystl growth nd ggregtion in humn urine suggesting tht it my interfere with the erly stges of stone formtion nd my represent n lterntive tretment of urolithisis (Sighinolfi et l., ). Further studies hve reveled the ntihypertensive (Iikuz et l., ), the ntioxidnt nd heptoprotective potentil of this medicinl plnt (Hrish nd Shivnndpp, ). These therpeutic properties hve een linked to the presence of ctive phytochemicls such s flvonoids, lkloids, terpenoids, lignns, tnnins, coumrins nd sponins from vrious prts of P. niruri L. (Bglkotkr et l., ). However, the potentil of this medicinl plnt ginst humn pthogens such s H. pylori still remins unknown. The possiility of comined therpy including phenolic phytochemicl-linked medicinl plnts nd lctic cid cteri with proiotic potentil on H. pylori inhiition my constitute novel nd low cost strtegies Received April Revised August Accepted July

2 9 L. G. RANILLA ET AL. towrds the solution to H. pylori-ssocited peptic ulcers nd ctive chronic gstritis s well s their long term impcts. Proiotic cteri re defined s live microorgnisms, which when dministered in dequte mounts, confer helth enefit on the host. Proiotic ctivity hs een ssocited with Lctocilli, Bifidocteri, Streptococcus, Enterococcus, non pthogenic E. coli nd Scchromyces oulrdii (FAO/OMS, ). It hs een shown tht proiotic cteri prevent infection of pthogenic cteri oth through ctivtion of the host s immune system nd through direct competition of the proiotic cteri with the pthogen. There is good evidence tht H. pylori is killed y lctocilli oth in vitro nd to limited extent in vivo (Zou et l., 9). Bsed on the ove rtionle, the ojective of this reserch ws to evlute the potentil of wter extrcts of Chncpiedr (Phyllnthus niruri L.) from Peru nd Ecudor for ntimicroil ctivity ginst Helicocter pylori nd different strins of lctic cid cteri with proiotic potentil such s Lctocillus cidophilus, Lctocillus csei nd Lctocillus plntrum.further, preliminry investigtions into the mode of ction of high phenolic wter extrcts of P. niruri L. on H. pylori inhiition were evluted sed on the rtionle tht certin simple phenolics could e proline nlog mimics nd my regulte ntimicroil ctivity through inhiition of proline oxidtion vi proline dehydrogense (Shetty nd Lin, ). MATERIALS AND METHODS Mterils. The commercil product from Ecudor which included dried leves of Phyllnthus niruri L. (Euphoricee) (rnd L Cholit ) ws purchsed from n Ecudorin Store in Hdley, MA (USA). Commercilly ville fresh leves of Phyllnthus niruri L. were purchsed in locl mrket in Lms-Sn Mrtin (Peru) (High Amzon) in Jnury, nd then dehydrted t C in hot ir oven until constnt weight. Specimens were uthenticted sed on comprisons to similr specimens confirmed previously y Rutter (99) nd Meji nd Renjifo (). Unless noted, ll chemicls were purchsed from Sigm Chemicl Co. (St Louis, MO). Strins used. Helicocter pylori (strin ATCC 9, which originted from humn gstric smples) ws otined from the Americn Type Culture Collection (Rockville, MD). The lctic cid cteri strins used in this study were the following: Lctocillus ulgricus ws isolted in previous study (Apostolidis et l., ). Lctocillus cidophilus ws supplied y Rosell Institute Inc. Montrel, Cnd (Lot#: XA, Seq#:), Lctocillus plntrum () (ATCC 99), Lctocillus plntrum () (NRRL B9), Lctocillus csei () (ATCC, PLP-XYL) nd Lctocillus csei () (PLP-). Extrct preprtion. A totl of g of powdered dried smple ws dded to ml of distilled wter nd refluxed t 9 C for min nd cooled. The extrcts were then filtered through filter pper (Whtmn No. ) nd mde up to ml with distilled wter ( mg/ml of smple concentrtion). The ph of the queous extrcts were corrected to nd centrifuged t 9 g for min. An liquot of the superntnt ws re-centrifuged t rpm for min efore ech ssy. Totl phenolics ssy. The totl phenolics were determined y the stndrd Folin-Cioclteu method s modified y Shetty et l. (99). Briefly, ml of diluted extrct ( mg/ml) ws trnsferred into test tue nd mixed with ml of 9% ethnol nd ml of distilled wter. To ech smple. ml of % (vol/vol) Folin-Cioclteu regent ws dded nd mixed. After min, ml of % N CO ws dded to the rection mixture nd llowed to stnd for min. The sornce ws red t nm. The stndrd curve ws estlished using vrious concentrtions of gllic cid in 9% ethnol, nd the results were expressed s mg of gllic cid equivlent per grm of smple dry weight (dw). Antioxidnt ctivity y,-diphenyl--picrylhydrzyl rdicl (DPPH) inhiition ssy. The stndrd DPPH scvenging ctivity ws determined y n ssy modified y Kwon et l. (). To. ml of mm DPPH in 9% ethnol, ml of diluted extrct ( mg/ml) ws dded, nd the decrese in sornce ws monitored fter min t nm (As extrct ). The sornce of control (distilled wter insted of smple extrct) ws lso recorded fter min t the sme wvelength (As control ). Therefore, the percentge of inhiition ws clculted y: ð % Inhiition ¼ As controlþ ðas extrct Þ ð Þ As control High performnce liquid chromtogrphy (HPLC) nlysis of phenolic profiles. The extrcts ( ml) were filtered through. mm filter. A volume of ml of extrct ws injected using n Agilent ALS utosmpler into n Agilent series HPLC (Agilent Technologies, Plo Alto, CA) equipped with DAD diode rry detector. The solvents used for grdient elution were (A) mm phosphoric cid (ph.) nd (B) % methnol. The methnol concentrtion ws incresed to % for the first min nd to % over the next min, then decresed to % for the next min nd ws mintined for the next min (totl run time, min). The nlyticl column used ws Agilent Zorx SB-C,. mm i.d., with pcking mteril of mm prticle size t flow rte of ml/min t room temperture. During ech run the sornce ws recorded t nm nd nm nd the chromtogrm ws integrted using n Agilent Chemsttion enhnced integrtor. Pure stndrds of chlorogenic cid, gllic cid nd ellgic cid in % methnol were used to clirte the stndrd curves nd retention times. Preprtion of strter culture of Helicocter pylori. Helicocter pylori ws cultured ccording to Stevenson et l. (). Stndrd plting medium (H. pylori gr pltes) were prepred y using g of specil peptone (Oxoid Ltd, Bsingstoke, Englnd) per liter, g of grnulted gr (Difco Lortories, Becton, Dickinson nd Co., Sprks, MD) per liter, g of sodium chloride Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()

3 PHYLLANTHUS NIRURI L. AND ANTIMICROBIAL ACTIVITY 9 (EM Science, Gistown, NJ) per liter, g of yest extrct (Difco) per liter, g of eef extrct (Difco) per liter of wter. Broth medi were prepred y g of specil peptone (Oxoid Ltd, Bsingstoke, Englnd) per liter, g of sodium chloride (EM Science, Gistown, NJ) per liter, g of yest extrct (Difco) per liter, g of eef extrct (Difco) per liter of wter. A volume of ml of stock H. pylori ws dded into test tues contining ml of sterile roth medi. They were incuted t C for h efore eing used for inoculting y the spred plte technique. The ctive culture ws then spred on H. pylori gr pltes to mke cteril lwn for the gr-diffusion ssy. Agr-diffusion ssy. The ntimicroil ctivity of smple extrcts ws nlysed y the gr-diffusion method. The ssy ws done septiclly using sterile. mm dimeter pper disks (Schleicher & Schuell, Inc., Keene, NH) to which ml of test extrcts were dded. Sturted disks were plced onto the surfce of seeded gr pltes. Controls consisted of disks with distilled wter only. Treted pltes were incuted t C for h in BBL GsPk jrs (Becton, Dickinson nd Co., Sprks, MD) with BD GsPk Cmpy continer system schets (Becton, Dickinson nd Co., Sprks, MD). The dimeter of the cler zone (no growth) surrounding ech disk ws mesured nd the zone of inhiition ws determined nd expressed in mm. For the dose dependency studies, the smple extrct ( mg/ml) ws diluted to, nd mg/ml with distilled wter. The ntimicroil ctivity of Phyllnthus niruri L.-ssocited pure phenolic compounds such s chlorogenic cid, ellgic cid nd gllic cid ws lso evluted t two concentrtions:. mg/disk nd mg/ disk. Ech experiment ws repeted two times nd consisted of three replictes ech time (three disks per smple or tretment). Proline growth response ssy. A model for the mode of ction of phenolic phytochemicls ws developed sed on the rtionle tht simple mono-phenolics in phytochemicl profiles could ehve s proline nlogs or proline nlog mimics nd re likely to inhiit proline oxidtion vi proline dehydrogense (Shetty nd Whlqvist, ). Further, the likely inhiitory effects of phenolic phytochemicls should e overcome y proline if the site of ction is proline dehydrogense. Bcteril lwns of H. pylori were prepred s descried previously. Plting medi were prepred y using stndrd plting medium s descried in the gr-diffusion ssy with some modifictions. Proline (Sigm, Louis, MO) ws dded into the medium to finl concentrtion of.,, nd mm). Different concentrtions of smple extrcts (,, nd mg/ml) were dded to pper disks ( ml) using micropipette. Sturted disks were plced onto surfce of seeded gr pltes. Pltes were incuted t C for h in BBL GsPk jrs (Becton, Dickinson nd Co., Sprks, MD) with BD GsPk EZ Cmpy continer system schets (Becton, Dickinson nd Co., Sprks, MD). The dimeter of the inhiition zone surrounding ech disk ws mesured (mm) nd the zone of inhiition ws determined. Lctic cid cteri prolifertion ssy. Initilly ml of frozen stock from the lctic cid cteril strins ws inoculted into ml MRS roth (Difco) nd incuted for h t C. Then, ml of the h grown strin ws re-inoculted into ml MRS roth for h t C. Smple extrcts ( mg/ml of concentrtion) were filter sterilized using sterile filters Millex GP. mm (Millipore Corportion, Bedford, MA). Filter sterilized smple extrcts ( ml) nd ml of the h grown strin (diluted times with sterile distilled wter) were dded into 9 ml of MRS roth tues nd incuted t C for h. A control with ml of sterile distilled wter insted of smple extrct ws lso included. An initil evlution of the lctic cid cteri prolifertion ws crried out only in MRS roth (Difco) y mesuring the opticl density t nm of the resuspended cells in ml of distilled wter ( ml liquot previously centrifuged t g for min) fter,, 9, nd h of growth. Bsed on the results from the previous evlution, the experiment ws repeted using the plte counting technique. Filter sterilized smple extrcts (dose of mg/ml in finl medium reflecting plte ssy exposure of Helicocter pylori) nd ml of the h grown strin (diluted times with sterile distilled wter) were dded into 9 ml of MRS roth tues nd incuted t C for h. A control with ml of sterile distilled wter insted of the smple extrct ws lso included. A volume of ml from the pproprite dilution ws plted on MRS gr (Difco) nd incuted in neroic BBL GsPk jrs (Becton, Dickinson nd Co., Sprks, MD) with BD GsPk EZ neroe continer system schets (Becton, Dickinson nd Co., Sprks, MD) t C for h to determine the CFU/ ml. The plte counting ws done t time nd fter nd h of cteri growth. Sttisticl nlysis. All experiments were performed y duplicte nd the nlysis t every time point from ech experiment ws crried out in triplicte (n = ). In the cse of HPLC nlysis, the experiments were performed t lest in triplictes. Results were expressed s men stndrd devition. Dt were sujected to pired Student s t-test nd - or -wy nlysis of vrince (ANOVA) with the Tukey s test (p <.) for men comprisons using the Sttgrphics Centurion XV softwre pckge (SttPoint Inc., Rockville, MD). RESULTS AND DISCUSSION Totl phenolics, ntioxidnt ctivity y DPPH nd HPLC nlysis Tle shows the totl phenolic contents of wter extrcts of Chncpiedr (Phyllnthus niruri L.) from different origins (Ecudor nd Peru) correlted to their free rdicl scvenging linked-ntioxidnt ctivity mesured y the DPPH ssy. The totl phenolic contents were higher in the Peruvin extrct thn in the Ecudorin extrct ( nd mg/g dw, respectively; p <.). However, the ntioxidnt ctivity ws quite similr in oth extrcts (9%). Specific phenolic compounds were detected nd quntified y HPLC-DAD (Tle ) nd the chromtogrms of oth Peruvin nd Ecudorin wter extrcts Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()

4 9 L. G. RANILLA ET AL. Tle. Totl phenolics nd DPPH scvenging ctivity of wter extrcts of Chncpiedr (Phyllnthus niruri L.) from Ecudor nd Peru Origin Totl phenolics (mg gllic cid/g smple dw) DPPH scvenging ctivity (%) Ecudor.9. Peru.9. Vlues re men SD; columns with different letters indicte sttisticlly significnt differences (p <., n = ). Tle. Phenolic composition of wter extrcts of Chncpiedr (Phyllnthus niruri L.) mesured y high performnce liquid chromtogrphy-diode rry detector Origin Phenolic compound (mg/g dw) Hydroxycinnmic cid Gllic cid Ellgic cid Ecudor Peru.. n.d... Vlues re men SD; expressed s chlorogenic cid; n.d., not detected. Columns with different letters indicte sttisticlly significnt differences (p <., n = ). re shown in Fig.. Hydroxycinnmic cid derivtives nd likely cffeoyl derivtives due to the similrity of their UV spectr to tht showed y chlorogenic cid were detected in oth extrcts, lthough the content in the Peruvin extrct ws significntly higher thn in the Ecudorin extrct (.. nd.9. mg/g dw expressed s chlorogenic cid, respectively). In ddition, Peruvin nd Ecudorin extrcts of P. niruri L. hd similr contents of ellgic cid (Tle ), ut gllic cid ws detected only in the Ecudorin wter extrct (.9. mg/g dw). These differences indicte the importnt effect of the smple origin on phenolic profiles of P. niruri L wter extrcts. Flvonoids such s quercetin, rutin, quercitrin, strglin nd ctechin hve een identified in extrcts from P. niruri L. (Bglkotkr et l., ). Methyl revifolin croxylte, coumrin derivtive, ws lso isolted from leves of P. niruri L. nd showed vsorelxnt effect on rt ortic rings (Iikuz et l., ). Among the polymeric molecules, ellgitnnins such s gerniin, repndusinic cid nd corilgin hve een lso found in extrcts of P. niruri L. (Bglkotkr et l., ). According to results from this study, neither flvonoids nor ellgitnnins were identified in queous extrcts of P. niruri L. under the conditions of the conducted experiments. However, the presence of hydroxycinnmic cids likely cffeoyl derivtives ws recently reported y our lortory in smple from Ecudor (Rnill et l., ). Ellgic cid itself is not thought to e nturlly present in plnts. Insted, polymers of gllic cid nd hexhydroxydiphenoyl (HHDP) re linked to glucose centres to form clss of compounds known s ellgitnnins (hydrolysle tnnins) (Rice-Evns et l., 99). When two gllic cid groups ecome linked side-y-side within tnnin molecule, the HHDP group is formed. After hydrolysis, the HHDP cid is dehydrted followed y spontneous lctoniztion forming ellgic cid (Aguiler-Cro et l., ). Furthermore, gllotnnins, the other group of hydrolysle tnnins, yield glucose nd gllic cid upon hydrolysis (Puupponen-Pimiä et l., ). This rtionle would Ecudorin extrct Asornce ( nm) Peruvin extrct Time (min) Figure. Chromtogrms of wter extrcts of Chncpiedr (Phyllnthus niruri L.), gllic cid;, hydroxycinnmic cid;, ellgic cid. Pek res were integrted t nm for quntifiction of detected phenolic compounds. Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()

5 PHYLLANTHUS NIRURI L. AND ANTIMICROBIAL ACTIVITY 9 explin the presence of ellgic cid nd gllic cid in wter extrcts of Chncpiedr likely derived from ellgitnnins or gllotnnins prtilly hydrolysed fter the hot wter extrction undertken in this study. Anticteril ctivity ginst Helicocter pylori nd proline growth response Both wter extrcts of Chncpiedr (Phyllnthus niruri L.) from Ecudor nd Peru showed similr nti-helicocter pylori ctivities nd dose-dependency trend ws oserved when four different concentrtions of smple were tested (,, nd mg of dried smple/ml) (Figs nd ). In order to determine if Chncpiedr linked-specific phenolics detected y HPLC-DAD re involved on its nti-h. pylori ctivity, the ntimicroil effect of pure phenolic compounds such s ellgic cid, gllic cid nd chlorogenic cid ws lso evluted y the grdiffusion ssy t two concentrtions (. nd mg/ disk). However, no inhiition ws oserved (dt not shown) in ll cses. Previous studies hve provided clues in niml cell nd cteri model systems tht phenolics could modulte cellulr redox response through the prolinessocited pentose phosphte pthwy (Shetty nd Whlqvist, ; Lin et l., ). Therefore, the rtionle for the proline growth response ssy ws to evlute if simple mono-phenolics in extrcts of Chncpiedr ehve s proline nlogs or proline mimics nd if so, could they inhiit proline oxidtion vi proline dehydrogense (PDH) t the plsm memrne level in prokryotic cell disrupting the oxidtive phosphoryltion linked-proton motive force (PMF) resulting in inhiition of the cterium (Kwon et l., ; Lin et l., ). If this is the cse then the ddition of proline could overcome the inhiition of proline nlog type phenolics with romtic ring structure (Shetty nd Lin, ). The results otined in the present study indicte tht the nti-h. pylori ctivity of wter extrcts of Chncpiedr is not ssocited with the proline dehydrogensesed oxidtive phosphoryltion inhiition. The ddition of proline into the H. pylori gr medi could not reverse the strong inhiitory ctivity of the extrcts even when higher doses of proline were tested (Fig. ). Therefore, mechnisms involving memrne relted rekdown or ltertions linked to iphenolics nd polymeric phenolics not detected y HPLC my e responsile for the nti-h. pylori ctivity of P. niruri L. extrcts y using different mechnisms of ction. Ellgitnnin-rich plnt extrcts hve een shown to possess inhiitory ctivity ginst multiple ntiioticresistnt H. pylori strins. Vorvuthikunchi nd Mitchell () using in vitro ssys reported tht oth queous nd ethnol extrcts from Punic grntum exhiited high ctivity ginst different strins of H. pylori. Previous studies hve reported tht Punic grntum fruits re rich in ellgitnnins such s puniclgin which lso hd potent inhiitory effect on methicillin-resistnt Stphylococcus ureus in vitro (Mchdo et l., ). Hydrolysle tnnins hve higher wter soluility thn other compounds nd this property my e importnt for their promising nticteril ctivities ginst H. pylori in vitro. Monomeric hydrolysle tnnins including ellgitnnins such s gerniin nd corilgin reveled especilly stronger nti-h. pylori ctivity thn oligomeric hydrolysle tnnins (Funtogw et l., ). During the cteril killing process, monomeric ellgitnnins rpidly ggregte H. pylori cells into clusters nd fuse them, which suggests tht these compounds ct on the memrne or memrne-linked surfce structures of H. pylori (Funtogw et l., ). Further, monomeric ellgitnnins exhiited dose-dependent memrne dmging ctivity nd possily this property contriute to their nti-h. pylori ction (Funtogw et l., ). Another possile mechnism of the nticteril ction of phenolics is ssocited to the cteril ntidhesion ctivity. Adhesion of cteri is vitl prerequisite for successful microil coloniztion nd infection. High moleculr-weight constituents of crnerry juice hve een shown to inhiit dhesion of H. pylori to immoilized humn mucus, erythrocytes nd cultured gstric epithelil cells (Burger et l., ). It ws suggested tht ellgitnnins rich crnerry juice my lso inhiit the dhesion of cteri to the stomch in vivo, nd my prove useful for the prevention of stomch ulcer tht is cused y H. pylori (Burger et l., ) or for the erdiction of H. pylori in comintion with proton pump inhiitors in ptients receiving tretment with conventionl triple therpy (Shmuely et l., ). Bsed on the ove rtionle, the strong nti-h. pylori ctivity found in wter extrcts of Chncpiedr my e ttriuted to the presence of ellgitnnins such s gerniin or corilgin, compounds tht hve een previously identified in the leves of Phyllnthus niruri L. (Bglkotkr et l., ). Most likely, these ellgitnnins were prtilly hydrolysed fter the hot wter extrction resulting in the formtion of ellgic cid, gllic cid nd other ellgitnnin derivtives. However, further studies re needed to confirm this premise. ) ) Figure. Helicocter pylori inhiition of the sme Chncpiedr (Phyllnthus niruri L.) wter extrct t different doses of smple concentrtion: (), nd mg/ml; (),, nd mg/ml. C, distilled wter s control. This figure is ville in colour online t wileyonlinelirry.com/journl/ptr Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()

6 9 L. G. RANILLA ET AL. Dimeter of the Inhiition Zone (mm) Dimeter of the Inhiition Zone (mm) mg/ml mg/ml mg/ml mg/ml Ecudorin extrct control. mm mm mm mm d c Ecudorin extrct mg/ml mg/ml mg/ml mg/ml c c c Peruvin extrct control. mm mm mm mm Proline concentrtion Figure. Effect of different concentrtions of Chncpiedr extrcts (Phyllnthus niruri L.) (mg/ml) with proline (.,, nd mm) on nti-helicocter pylori ctivity. Control, without proline. Brs with different letters t the sme extrct concentrtion indicte sttisticlly significnt differences (p <., n = )., no significnt differences t the sme extrct concentrtion (p >.). d c Peruvin extrct Figure. Anti-Helicocter pylori ctivity of two wter extrcts of Chncpiedr (Phyllnthus niruri L.) from Ecudor nd Peru t four different doses of smple concentrtion. Brs with the sme letters t the sme extrct concentrtion indicte no significnt differences (p >.). Brs with different letters t ech smple origin dt (Ecudorin or Peruvin) indicte sttisticlly significnt differences (p <., n =). As mentioned ove, the totl phenolic contents nd the HPLC phenolic profiles of P niruri L. extrcts vried ccording to the origin. However, extrcts from Ecudor nd Peru did not hve significnt differences in their free rdicl scvenging-linked ntioxidnt ctivities (Tle ) nd proly this chrcteristic could e lso correlted to their similr nti-h. pylori ctivities. Solule phenolics hve een suggested to exert their ntimicroil effect y cusing hypercidifiction vi proton dontion t the plsm memrne interfce of the microorgnism nd intrcellulr cytosolic cidifiction, excess of which cn disrupt H + -ATPse required for ATP synthesis (Kwon et l., ). The inhiition of H. pylori y P. niruri L. wter extrcts could e due to the proton donting effect of solule phenolic ntioxidnt compounds which my inhiit cytosolic dehydrogenses when cidifiction is high. Further, phenolics my hve the ility to quench free electrons from the electron trnsport chin long the cteril memrne or inhiit dehydrogenses linked proton efflux. This could disrupt the flow of the electrons t the level of cytochromes nd inhiit the growth of cteri y disrupting the protons required for oxidtive phosphoryltion (Shetty nd Whlqvist, ). Ellgitnnins hve een shown to possess strong scvenging properties ginst free rdicls indicting their potentil ility to donte protons (Reddy et l., ). This provides clues tht they could modulte interfcil nd cytosolic cidity which could e inhiitory if not countered y dehydrogenses. P. niruri L. hs een reported to contin lkloids, terpenes, lignns, sponins, esides phenolic compounds such s ellgitnnins nd flvonoids (Bglkotkr et l., ). The nti- H. pylori ctivity of lignns hs een reported previously (Toyod et l., ). Thus, inhiition of H. pylori y wter extrcts of Chncpiedr my e likely ssocited to the synergistic ction of such compounds in comintion with ellgitnnins nd other solule phenolic derivtives. P. niruri L. hs demonstrted nticteril ctions ginst Stphylococcus, Micrococcus nd Psteurell cteri (Veermuthu et l., ). However, the potentil of this plnt for inhiiting the gstric cncer linked-h. pylori is reported for the first time in this study. Effect on proiotic lctic cid cteri prolifertion An initil evlution of the effect of Chncpiedr (Phyllnthus niruri L.) wter extrcts on the prolifertion of lctic cid cteri with proiotic potentil ws crried out y mesuring the cteril cell concentrtion t nm fter,, 9, nd h of growth in MRS roth (Fig. ). A non-proiotic microorgnism (Lctocillus ulgricus) ws lso included in the screening step. Overll, the ddition of wter extrcts of P. niruri L. ( mg/ml of smple concentrtion into the MRS roth, reflecting plte ssy exposure of Helicocter pylori), independent of the smple origin, did not ffect the growth of Lctocillus cidophilus, Lctocillus csei nd, nd Lctocillus plntrum nd when compred with the control (distilled wter). Sttisticlly, some differences were oserved t h of growth (p <.). Therefore, the experiment ws repeted y using the more ccurte plte counting technique t nd fter h of growth in liquid medi Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()

7 PHYLLANTHUS NIRURI L. AND ANTIMICROBIAL ACTIVITY 9 Ecudorin extrct Peruvin extrct Control (distilled wter) L. cidophilus L. ulgricus 9 9 Asornce ( nm) L. csei 9 L. csei 9 c L. plntrum c L. plntrum 9 9 Figure. Evlution of the effect of Chncpiedr (Phyllnthus niruri L.) wter extrcts on lctic cid cteri prolifertion (sornce t nm). Brs with different letters t the sme time of incution indicte sttisticlly significnt differences (p <., n = );, no significnt differences (p >.). (MRS roth). As shown in Fig., the growth of evluted Lctocillus strins ws not inhiited y ny of the P. niruri L. extrcts ( mg/ml of smple concentrtion into the MRS roth) (p >.). Similr to the ove studies, Puupponen-Pimiä et l. () using the liquid culture technique, oserved tht the ellgitnnin rich-rsperry extrct did not inhiit the growth of Lctocillus prcsei, Lctocillus rhmnosus nd Bifidocterium lctis t low concentrtions ( mg/ml). However, when five times higher concentrtion of rsperry extrct ws used, the growth of tested Lctocillus strins ws clerly inhiited. The sme uthors lso reported tht growth of selected Grm-negtive cteri such s Slmonell nd some E. coli strins were generlly inhiited y high phenolicextrcts from common Finnish erries, wheres no inhiition ws oserved ginst Grm-positive lctic cid cteri. They hypothesized tht oserved vritions my reflect differences in cell surfce structures etween Grm-negtive nd Grm-positive cteri. In prticulr, the outer memrne of Grm-negtive cteri functions s preventive rrier ginst hydrophoic compounds. Funtogw et l.() using in vitro ssys, indicted tht monomeric ellgitnnins did not hve nticteril ctivity ginst non-pthogenic E. coli ( norml inhitnt of the humn intestinl trct), suggesting tht hydrolysle tnnins my e le to inhiit H. pylori without ffecting potentilly eneficil nd well dpted intestinl cteril flor. Further, the sme uthors highlighted the nti-h pylorictivity of hydrolysle tnnins without ffecting the viility of gstric epithelil cells. In ddition, similr to the current results, recent study from our lortory lso showed the potentil of polyphenol rich-te extrcts ginst H. pylori nd the lck of inhiition of selected eneficil lctic cid cteri. Besides the possile effect due to differences in cellulr wll configurtion, differences in metolism linked to Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()

8 9 L. G. RANILLA ET AL. Log CFU/mL 9 L. cidophilus 9 L. csei Ecudorin extrct Peruvin extrct Control (distilled wter) 9 L. csei 9 L. plntrum 9 L. plntrum Figure. Effect of Chncpiedr (Phyllnthus niruri L.) wter extrcts on lctic cid cteri prolifertion (CFU/mL). Brs with different letters t the sme time of incution indicte sttisticlly significnt differences mong smples (p <., n = );, no significnt differences (p >.). energy genertion (prtil oxidtive phosphoryltion in microerophilic cteri such s H. pylori in contrst to sustrte level phosphoryltion in lctic cid cteri) my lso e involved (Ankolekr et l., ). New therpies pplied for the tretment of H. pylori infection suppress not only H. pylori ut lso intestinl cteril flor, which cuses side effects such s dominl pin nd dirrhe (Wermeille et l., ). In this study, the wter extrcts of P. niruri L. inhiited H. pylori ut t the sme dose the growth of evluted lctic cid cteril strins with proiotic potentil ws not inhiited. This shows the promise of Chncpiedr extrcts for potentil tretment or prevention of H. pylori infections without likely cusing side effects linked to inhiition of eneficil intestinl proiotic cteri. The chllenge of overcoming Helicocter pylori ntiiotic resistnce hs focused reserch on the isoltion nd chrcteriztion of new ntimicroil compounds from vriety of nturl sources including medicinl plnts. In this study, wter extrcts of the medicinl plnt Chncpiedr (Phyllnthus niruri L.) from Ecudor nd Peru inhiited the humn pthogen H. pylori in dose dependent mnner. The totl phenolic contents ( nd mg/g dw, for Peruvin nd Ecudorin extrct, respectively) nd the HPLC phenolic profiles of ech extrct vried ccording to the smple origin. However, the free rdicl scvenging linked-ntioxidnt ctivity ssessed y the DPPH method ws high nd similr in oth extrcts (9%). The mjor phenolic compounds were ellgic cid nd hydroxycinnmic cid derivtives in oth extrcts, ut gllic cid ws detected only in the Ecudorin wter extrct. Insights into the mode of ction of Chncpiedrlinked phenolics on H. pylori inhiition indicted tht the nti-h. pylori ctivity of this plnt my not e vi proline dehydrogense-sed oxidtive phosphoryltion inhiition. Therefore, solule polymeric phenolics such s ellgitnnins or other non-phenolic compounds re proly involved nd my ct in synergistic mnner, potentilly ffecting memrne coupled energy function. Independent of the smple origin, lctic cid cteri with proiotic Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()

9 PHYLLANTHUS NIRURI L. AND ANTIMICROBIAL ACTIVITY 99 potentil such s Lctocillus cidophilus, Lctocillus plntrum nd Lctocillus csei were not inhiited y Chncpiedr wter extrcts t dose of mg/ml, which inhiited H. pylori. These results suggest tht Chncpiedr could e used potentilly for the prevention or tretment of H. pylori infections with the dditionl dvntge of not ffecting eneficil intestinl proiotic lctic cid cteri. However, reserch on the elucidtion of the exct mechnism for the nti-h. pylori ctivity of Chncpiedr nd further in vivo studies on the eneficil effects re needed. Conflict of Interest The uthors hve declred tht there is no conflict of interest. REFERENCES Aguiler-Cro A, Augur C, Prdo-Brrgn LA, Fvel-Torres E, Aguilr CN.. Microil reduction of ellgic cid nd iodegrdtion of ellgitnnins. Appl Microiol Biotechnol : Ankolekr C, Johnson D, Pinto MDS, Johnson K, Le R, Shetty K.. 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The diverse ntioxidnt systems of Helicocter pylori. Mol Microiol :. Wermeille J, Cunninghm M, Dederding JP et l.. Filure of Helicocter pylori erdiction: is poor complince the min cuse? Gstroenterol Clin Biol : 9. Zou J, Dong J, Yu XF. 9. Met-nlysis: Lctocillus contining qudrupole therpy versus stndrd triple first-line therpy for Helicocter pylori erdiction. Helicocter : 9. Copyright John Wiley & Sons, Ltd. Phytother. Res. : 9 99 ()